The major goal of this research is to elucidate the substrate specificity and mechanism of action of lecithin cholesterol acyltransferase (LCAT), the enzyme responsible for the synthesis of extracellular cholesterol esters in human plasma. During the past grant period we prepared discoidal HDL analogs of various compositions and investigated their reactions with LCAT. We showed that all HDL apolipoproteins are capable of activating LCAT, but that a specific three-dimensional structure of apo A-I is required for optimal activation. In addition, we demonstrated that the enzyme activity is sensitive to the phosphatidylcholine (PG) chain length, unsaturation, and concentration in the interface, as well as to the anion composition of the medium. On the other hand, we determined that LCAT activity is not a function of the phase state of the lipids, of the cholesterol concentration, nor of the PC/cholesterol ratios in the substrate particles. We propose to investigate further the substrate selectivity and mechanism of the LCAT reaction by examining the interaction of the enzyme with various substrate particles, and determining the intrinsic kinetic constants from the kinetic and equilibrium binding results. This work will involve the preparation of milligram amounts of the enzyme, its fluorescence labeling, characterization of the enzyme structure in the presence and absence of various effectors, binding studies of LCAT with discoidal substrates, speherical HDL analogs of various sizes, and native subfractionated HDL. In addition, we will study the phospholipase reaction of LCAT with mixed detergent-lipid micelles, small PC micelles, and monomeric lipids and esters, in order to gain a better understanding of the catalytic step, and the interface and activator apolipoprotein roles in the enzymatic reaction.